How to stop the glutaraldehyde crosslinking before analysis in FPLC. For SDS PAGE we add sample buffer after cross-linking to stop the reaction but for FPLC what can I do?
Glutaraldehyde can be quenched through the addition of primary amine-containing buffers such as Tris. So before injecting your sample into the FPLC, add 5-10mM (final concentration) Tris at your desired pH and let it incubate for a few minutes. This will quench the cross-linker completely and make it safe for FPLC analysis.
Here is a paper that uses this method of quenching through the molecular excess of Glycine - another suitable quenching agent Article Structure of Glutaraldehyde Cross-Linked Ryanodine Receptor
You can also add NaBH4 to change the aldehyde groups to hydroxyl groups
Dear Sunita, thank you for sharing this very interesting technical question with the RG community. If you have sodium borohydride (NaBH4) available in your lab, you can try this reagent. For a detailed experimental procedure for preparing a quenching buffer solution containing NaBH4 as quenching agent please have a look at the following useful reference (page 1226):
Effect of Glutaraldehyde Concentration on the Physical Properties
of Polymerized Hemoglobin-Based Oxygen Carriers
Article Effect of Glutaraldehyde Concentration on the Physical Prope...
It is interesting to note that reducing agents like NaBH4 and NaCNBH3 also reduce the Schiff-base units (–N=CH–) formed during the crosslinking reaction. However, this has no quenching effect as the links on the protein surface remain intact. For more infromation about this please see the following interesting article:
Glutaraldehyde Cross-Linking Increases the Stability of Lumbricus terrestris Erythrocruorin
Article Glutaraldehyde Cross-Linking Increases the Stability of Lumb...
Unfortunately both articles have not yet been posted as public full texts on RG. For the first article (if it is of interest to you) you could try to e-mail the senior author at [email protected] (address is publicly available in Open Access papers). The corresponding author of the second paper has an RG account (https://www.researchgate.net/profile/Jacob-Elmer) so that you can request the full text directly from him.
Good luck with your research work and best wishes, Frank Edelmann
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